As has been demonstrated in experimental in vitro data, pre-clinical animal models, and patient studies, vitamin B12 is a co-enzyme necessary in cell division, as well as cellular metabolism, in proliferating normal and neoplastic cells. Insufficient vitamin B12 causes cellular division to be held in abeyance and ultimately may result in apoptosis. The nutrient is generally derived from dietary intake and is transported throughout the body complexed to transport proteins. The complex of transport protein and vitamin B12 is recognized by a cellular receptor which internalizes the complex and releases the vitamin intracellularly. The overall process has been reviewed in GUT 31:59, 1991. Vitamin B12 is taken in through the diet. Binding proteins in the saliva (R-binder) and gut (intrinsic factor-(IF)) complex vitamin B12 after release from endogenous binding proteins by action of enzymes and low pH in the stomach. Vitamin B12 is transferred across the intestinal epithelium in a receptor specific fashion to transcobalamin II (TcII). The vitamin B12/transcobalamin II complex is then transported throughout the body and recognized by receptors present on dividing cells, internalized and released within the cell where it is utilized by certain enzymes as a co-factor.
The high affinity receptor in dividing tissues or cells responsible for internalization of vitamin B12 recognizes transcobalamin II complexed with vitamin B12. The vitamin B12/TcII receptor recognizes only the vitamin B12/TcII complex and not the serum transport protein or the vitamin alone. The receptor is down-regulated on non-dividing cells; the mechanism for supplying non-dividing cells with vitamin B12 is poorly understood. However, it is known that more vitamin B12 is required during cell division than during metabolism, and that the vitamin B12/TcII receptor is the only high affinity means for cellular uptake of vitamin B12 during cell division. When stimulated to divide, cells demonstrate a five to ten fold increase in transient expression of this receptor leading to vitamin B12 uptake which precedes actual DNA synthesis (J. Lab. Clin. Med. 103:70, 1984). Vitamin B12 receptor levels may be measured by binding of 57Co-vitamin B12 complexed to transcobalamin II (present in serum) on replicate cultures grown in chemically defined medium without serum. No receptor mediated uptake occurs in the absence of carrier protein.
Dividing cells, induced to differentiate, lose receptor expression and no longer take up vitamin B12. More importantly, leukemic cells, deprived of vitamin B12, will stop dividing and die (Acta Haemat. 81:61, 1989). In a typical experiment, leukemic cell cultures were deprived of serum for 3 days, and then supplemented either with serum (a source of vitamin B12) or a non-metabolizable analogue of vitamin B12 and cultured up to five days. Cell cultures supplemented with vitamin B12 continued to grow, whereas those deprived of the active nutrient stopped growing and die.
Based on these observations, it has been suggested that whole body deprivation of vitamin B12 may be useful in the treatment of cancer or other disorders characterized by uncontrolled growth of cells. Moreover, because of the critical role played by vitamin B12-containing enzymes in cell division, it is believed that vitamin B12 deprivation may be used in combination with chemotherapeutic drugs which inhibit cellular replication. For example, when vitamin B12 depletion was combined with methotrexate, the two modalities together were more efficient in depleting folate levels in leukemic cells than either alone (FASEB J. 4:1450, 1990; Arch. Biochem. Biophys. 270:729, 1989; Leukemia Research 15:165, 1991). Folates are precursors in the production of DNA and proteins. In typical experiments, cultures of leukemic cells were exposed to nitrous oxide for several hours to convert the active form of endogenous vitamin B12 to an inactive form. Replicate cultures were then left without further treatment, or additionally treated with methotrexate. Cellular folate levels were measured three days later. Cells treated with the combination (i.e., both methotrexate and inactive vitamin B12) showed a more striking decrease in cellular folate levels than with either of the two approaches alone. This combination also results in a higher cell kill in vitro. When this approach was applied to the treatment of highly aggressive leukemia/lymphoma in animal models (Am. J. Haematol. 34:128, 1990; Anticancer Res. 6:737, 1986; Cancer Chemother. Pharmacol. 17:114, 1986; Br. J. Cancer 50:793, 1984), additive or synergy of anti-tumor action was observed, resulting in prolonged remissions and cures. The following Table 1 summarizes the observed additive or synergistic results:
TABLE 1Vitamin B12 Depletion (Nitrous Oxide) in Combination TherapyDrugs Used inCombinationwith Vitamin B12TherapeuticStudyDepletionResultsMyelocytic leukemia/ratscycloleucineadditive5-FUadditivemethotrexatesynergisticAcute leukemia/rats5-FUadditiveAcute leukemia/ratsmethotrexatesynergisticAcute leukemia/ratscycloleucinesynergistic
A key finding in the experiments described above was that short-term (hours to days), whole body depletion of vitamin B12 can act synergistically with chemotherapeutic drugs (such as methotrexate and 5-FU) to inhibit tumor growth and treat animals with leukemia/lymphoma. Despite synergistic anti-tumor activity, there was no toxicity attributable to the short-term vitamin B12 depletion for proliferating normal cells. This combination therapy was demonstrated in multiple animal models. Observations in patients have indicated that long-term (months to years) vitamin B12 depletion is required to produce significant normal tissue toxicity. Even in those cases, subsequent infusion of vitamin B12 can readily reverse symptomology (Br. J. Cancer 5:810, 1989).
Because of the promise of this therapeutic approach, various methods have been sought to efficiently and controllably perform a temporary depletion of vitamin B12. Such methods, however, affect all of the body's stores of vitamin B12. They include dietary restriction, high doses of vitamin B12 analogues (non-metabolizable-competitive antagonists which act as enzyme inhibitors), and nitrous oxide (transformation of vitamin B12 to inactivate form). These different methods have been used in culture systems and in animals to deplete vitamin B12. The most efficient and the most utilized method has been the inhalation of nitrous oxide (laughing gas). Animals are maintained typically under an atmosphere of 50% to 70% of nitrous oxide for periods from a few hours to a few days, causing the conversion of endogenous vitamin B12 into an inactive form. This methodology has been utilized in combination with drugs for therapy of leukemia/lymphoma. A further method for vitamin B12 depletion involves infusion of a non-metabolizable analogue of vitamin B12 which essentially dilutes out the active form. This form of therapy is not specific for dividing cells but affects liver dependent metabolic processes. Another approach includes restricting the dietary intake of vitamin B12. This method, however, requires very long periods of dietary restriction and is offset by hepatic storage of vitamin B12. All of these methods suffer from problems of specificity, since they affect both vitamin B12-dependent growth as well as basal metabolism, and therefore are not particularly suited to the development of anti-proliferative pharmaceutical products.
Accordingly, there is a need in the art for agents which will cause the cellular depletion of vitamin B12, and which selectively affect dividing cells. The present invention fulfills this need, and provides further related advantages.